Subobjective B: better characterize those bacterophagous eukaryotic microbes that may convey and help establish in produce pathogenic bacteria.

Objective 2: Develop a molecular phylogeny of coccidia in fish in order to better define their potential risk to food safety and security, and in order to better understand the relationship between Eimeriidae (including the agents of avian coccidiosis) and the Sarcocystidae (including the agent of human toxoplasmosis).

Objective 3: Better define the historical and ongoing interactions among wildlife and livestock reservoirs of Toxoplasma gondii through comparative population genetic analysis.

1b.Approach (from AD-416)
Several genes will be sequenced from parasites obtained from a wide array of animals, both domesticated and wild. These will be compared to each other, and to sequences obtained from human beings, in order to define the diversity and epidemiology of these parasite species. Homologues will be characterized for genes whose global variation has already begun to be studied in T. gondii, including the Intergenic Spacer sequence between rRNA genes, beta tubulin introns, and others. Characterization of microsatellite alleles will be considered as a second approach which, although requiring a greater investment of time and resources, should provide greater population genetic resolution than is possible with current loci.

3.Progress Report
In FY2010 we developed and applied high throughput genome sequencing approaches to discovering heritable differences among isolates of Trichinella, a zoonotic parasite of pork and used these methods to develop the first comprehensive assessment of the species of Trichinella most frequently encountered in the United States: Trichinella (T.) murrelli. This activity resulted in a publication describing new polymorphisms in the mitochondrial genome of T. spiralis and the submission of a manuscript which establishes the feasibility of using one parasite’s genome as a scaffold by which to assemble sequences of the other. We continued using genetic variation as a basis to better understand transmission patterns and evolutionary diversification in Toxoplasma gondii, the single greatest food-borne parasitic threat to human reproductive health. ARS Researchers in Beltsville, MD contributed to genome re-sequencing efforts, developed a new synthesis of population genetic patterns (identifying especially widespread dissemination of strains bearing certain chromosomal variants), and collected new genotypic data on U.S. isolates of the agent of toxoplasmosis.

ARS Researchers in Beltsville, MD attributed certain infections to known etiological agents (for example, a clinically unusual case of disseminated sarcocystosis) using molecular diagnostics and phylogenetic comparison, and used similar methods to establish other infections as new to science (for example, helping name a new species of Sarcocystis in conjunction with collaborators in Kunming, China).

With Hungarian and British collaborators, ARS Researchers in Beltsville, MD completed data collection and most of the data analysis on a broad survey of coccidian parasites of fish. These efforts have identified parasites which may have the ability to encyst in fish muscles, potentially serving as new food-borne agents. This work also forces a revision of long-held views of the diversity and origins of coccidian parasites (which include several zoonotic pathogens and many parasites that plague agricultural livestock).

ARS Researchers in Beltsville, MD helped establish that the agent of severe human malaria (Plasmodium falciparum) became established as a human pathogen relatively recently in our evolutionary history, and identified chimpanzees as the intermediate hosts to which this parasite had been previously restricted. Explaining the origins of one of humanity’s major scourges received considerable attention (it was published in the Proceedings of the National Academy of Sciences and was featured in Science, The New York Times, and CNN). The origins of this particular parasite in an Old World primate contrasts markedly with the origins of certain zoonotic food-borne parasites, which we have established relate to particular animal husbandry practices.

4.Accomplishments
1.
Agricultural practices can constrain or promote the spread of animal diseases, including diseases which can be transmitted via food consumption. Trichinella spiralis is one such zoonotic food-borne agent, which can cause human trichinellosis if swine are raised improperly. ARS Researchers at Beltsville, MD identified notably little variation in the mitochondrial genome of T. spiralis sampled from the Americas and from Europe, substantiating the idea that the parasite was brought to the Western Hemisphere by European farmers. ARS Researchers at Beltsville, MD identified numerous genetic features which distinguish T. spiralis (once common in pork) from T. murrelli (which remains prevalent in wild mammals in North America); meanwhile, they obtained additional evidence that the pork parasite represents a highly inbred lineage in the United States. This work will lay a foundation for future work to assess risks posed by either parasite to the safety of pasture-raised pork.

2.
Pregnant women, their fetuses, and persons with AIDS are vulnerable to infections with Toxoplasma gondii, which may be acquired from infected meat or from contact with the feces of cats. Although new parasite genotypes can result from sexual recombination in cats, actual surveys of parasites in people and animals repeatedly encounter certain over-represented strains. Working with an international team, ARS scientists at the Beltsville Agricultural Research Center developed a broader view of parasite diversity by defining the amount of variation in parasite genes, by assessing where particular genotypes occur, and by evaluating the importance of recombination in generating new parasite strains. These results suggest that the parasite endures prolonged intervals of genetic stasis, punctuated by occasional (but consequential) episodes of genetic re-assortment. In the future, this model will be subjected to additional scrutiny, and evaluated for its significance to zoonotic risk and clinical disease.

5.Significant Activities that Support Special Target Populations
The work on toxoplasmosis is especially important to the health of pregnant women, fetal and newborn health, and the health of persons afflicted by HIV-AIDS.